Dynamics of yeast plasma membrane domains

Plasmatic cellular mebrane consists of specialized domains which differ in shape, function and size. This compartmentalization allows cells to coordinate and separate a large number of diverse processes. Disorders of cell organization at the level of membrane domains are increasingly associated with the expansion of pathological disorders. Microdomains also play a role in the virulence of pathogenic organisms, e.g. some types of yeast. One of the best described microdomains of the yeast plasma membrane is the MCC (membrane compartment of arginine permease Can1), which is stabilized by a protein complex called the eisosome. The composition of the MCC/eisosome is described in detail, the dynamics of the individual components in the basal state (i.e. without stress) has not yet been described. The aim of this thesis is to summarize current knowledge about the dynamics of the proteins of this microdomain and its relationship with other domains of cell membranes through a detailed review of the available literature. And at the same time by using the advanced fluorescence method FRAP (fluorescence recovery after photobleaching) to show in my own experiment that in the basal state there is an exchange of individual Nce102 molecules in the MCC/eisosome microdomain. Key words: yeast, plasma membrane,...Plazmatická membrána buněk se skládá ze specializovaných domén, které se liší svým tvarem, funkcí a velikostí. Toto rozdělení umožňuje buňkám koordinovat a oddělit velké množství různorodých procesů. Poruchy organizace buňky na úrovni membránových domén je stále více spojováno se vznikem patologických poruch. Svou roli mají mikrodomény též ve virulenci patogenních organismů, např. některých druhů kvasinek. Jednou z nejlépe popsaných mikrodomén plazmatické membrány kvasinek je MCC (membránový kompartment argininové permeázy Can1), jenž je stabilizován proteinovým komplexem zvaným eisosome. Složení této mikrodomény je detailně popsáno, dynamika jednotlivých komponent v bazálním stavu (tj. bez stresu) dosud popsána nebyla. Cílem této práce je podrobným přehledem dostupné literatury shrnout současné poznatky o dynamice proteinů této mikrodomény a jejím vztahu s ostatními doménami buněčných membrán. Práce zároveň prostřednictvím pokročilé fluorescenční metody FRAP (česky obnova fluorescence po fotovybělení) na vlastním experimentu ukazuje, že v bazálním stavu dochází k výměně jednotlivých molekul Nce102 v mikrodoméně MCC/eisosom. Klíčová slova: kvasinky, plazmatická membrána, membránové domény, mikroskopie, FRAP, MCC, eisosom, Sur7, Nce102Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologiePřírodovědecká fakultaFaculty of Scienc

A recurrent mitochondrial p.Trp22Arg NDUFB3 variant causes a distinctive facial appearance, short stature and a mild biochemical and clinical phenotype

Background Isolated Complex I deficiency is the most common paediatric mitochondrial disease presentation, associated with poor prognosis and high mortality. Complex I comprises 44 structural subunits with at least 10 ancillary proteins; mutations in 29 of these have so far been associated with mitochondrial disease but there are limited genotype-phenotype correlations to guide clinicians to the correct genetic diagnosis. Methods Patients were analysed by whole-exome sequencing, targeted capture or candidate gene sequencing. Clinical phenotyping of affected individuals was performed. Results We identified a cohort of 10 patients from 8 families (7 families are of unrelated Irish ancestry) all of whom have short stature (C, p.Trp22Arg NDUFB3 variant. Two sibs presented with primary short stature without obvious metabolic dysfunction. Analysis of skeletal muscle from three patients confirmed a defect in Complex I assembly. Conclusions Our report highlights that the long-term prognosis related to the p.Trp22Arg NDUFB3 mutation can be good, even for some patients presenting in acute metabolic crisis with evidence of an isolated Complex I deficiency in muscle. Recognition of the distinctive facial features—particularly when associated with markers of mitochondrial dysfunction and/or Irish ancestry—should suggest screening for the p.Trp22Arg NDUFB3 mutation to establish a genetic diagnosis, circumventing the requirement of muscle biopsy to direct genetic investigations

Novel homozygous variants in PRORP expand the genotypic spectrum of combined oxidative phosphorylation deficiency 54

Biallelic hypomorphic variants in PRORP have been recently described as causing the autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a phenotypic spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy. Here, we report three additional families with homozygous missense PRORP variants with pleiotropic phenotypes. Each missense variant altered a highly conserved residue within the metallonuclease domain. In vitro mitochondrial tRNA processing assays with recombinant TRMT10C, SDR5C1 and PRORP indicated two COXPD54-associated PRORP variants, c.1159A>G (p.Thr387Ala) and c.1241C>T (p.Ala414Val), decreased pre-tRNAIle cleavage, consistent with both variants impacting tRNA processing. No significant decrease in tRNA processing was observed with PRORP c.1093T>C (p.Tyr365His), which was identified in an individual with leukodystrophy. These data provide independent evidence that PRORP variants are associated with COXPD54 and that the assessment of 5' leader mitochondrial tRNA processing is a valuable assay for the functional analysis and clinical interpretation of novel PRORP variants

OXA1L mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect

OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic OXA1L variants (c.500507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild-type human OXA1L in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or Drosophila melanogaster caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA-encoded subunits of complexes I, IV and V. Our data verify the pathogenicity of these OXA1L variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes.Peer reviewe

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals’ samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp−/− mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp−/− MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp(-/-) mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp(-/-) MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia.This study was supported by the German BMBF and Horizon2020 through E-Rare project GENOMIT (01GM1603 and 01GM1207 to H.P.; FWF-I 2741-B26 to J.A.M.); Vereinigung zur Förderung Pädiatrischer Forschung Salzburg; EU FP7 MEET Project (317433 to H.P. and J.A.M.); Horizon2020 Project SOUND (633974 to H.P.); Marie Skłodowska-Curie Actions Reintegration Fellowship (Mitobiopath-705560 to C.G.); UK NHS Highly Specialised Mitochondrial Service (R.W.T.); Wellcome Centre for Mitochondrial Research (203105/Z/16 to Z.M.C.-L., R.N.L., and R.W.T.); MRC Centre for Neuromuscular Diseases (G0601943 to R.W.T. and P.F.C.); Lily Foundation (R.W.T. and K.T.); UK NIHR fellowship (NIHR-HCS-D12-03-04 to C.L.A.); Wellcome Senior Fellowship (101876/Z/13/Z to P.F.C.); UK NIHR award and MRC Mitochondrial Biology Unit (MC_UP_1501/2 to P.F.C.); NIH (R01 GM0077465 and R35 GM122455 to V.K.M.); EMBO fellowship (ALTF 554-2015 to A.A.J.); UK MRC core funding for the Mitochondrial Biology Unit of the University of Cambridge (MC_U105697135 to A.R.D., P.R.G., and M. Minczuk); Portuguese Fundação para a Ciência e a Tecnologia (PD/BD/105750/2014 to P.R.G.); Italian Telethon (GSP16001 to G.P.C.); Fondazione Cariplo (2014-1010 to D.R.); Strategic Research Center in Private Universities from MEXT; and Practical Research Project for Rare/Intractable Diseases from AMED

Energy label of building containment

Předkládaná bakalářská práce popisuje metodiku výpočtu energetického štítku obálky budovy. Obsahuje samotný energetický štítek vytvořený pro konkrétní bytový dům ve stávajícím stavu i po navrhnutém opatření. Seznamuje s navrhnutými opatřeními a hodnotí stavbu z hlediska výpočtů a zároveň pořízených termovizních snímků.Katedra elektroenergetiky a ekologieObhájenoThis Bachelor thesis describes the methodology of calculation the energy label of building containment. It also contains the actual label created for a specific block of flats in the current state and even after suggested changes. In my Bachelor thesis are also introduced suggested changes and the building is evaluated from the perspective of calculations and thermo-visual footage

Evaluation of the Financial Situation of the Company and Proposals for its Improvement

The topic of this bachelor thesis is the evaluation of the financial situation of the company Víno Hruška s.r.o. for the period 2017 to 2021 with the help of financial analysis methods. The bachelor thesis is divided into three parts. The first part describes the theoretical basis of the thesis, which are further used in the practical part. The practical part is focused on the description and analysis of the company. The third part is the design part, which contains proposals for improving the financial situation of the company